In glutamatergic synapses, glutamate receptors (GluRs) associate with many other proteins involved in scaffolding and signal transduction. The ontogeny of these postsynaptic density (PSD) proteins involves changes in their composition during development, paralleling changes in GluR type and function. In the CA1 region of the hippocampus, at postnatal day 2 (P2), many synapses already have a distinct PSD. We used immunoblot analysis, subcellular fractionation, and quantitative immunogold electron microscopy to examine the distribution of PSD proteins during development of the hippocampus. Synapses at P2 contained substantial levels of NR1 and NR2B and most GluR-associated proteins, including SAP102, SynGAP, the chain of proteins from GluRs/SAP102 through GKAP/Shank/Homer and metabotropic glutamate receptors, and the adhesion factors, cadherin, catenin, neuroligin, and Nr-CAM. Development was marked by substantial decreases in NR2B and SAP102 and increases in NR2A, PSD-95, AMPA receptors, and CaMKII. Other components showed more moderate changes.

Subunit composition of NMDA receptors (NMDARs) determines a range of physiological properties, downstream signaling effects, and binding partners. Differential localization of NR2A- or NR2B-containing NMDARs within the neuron and subunit-specific protein associations may explain differences in NR2A and NR2B contributions to synaptic plasticity and excitotoxic cell death. This question is complicated by the existence of tri-heteromeric complexes (NR1/NR2A/NR2B). To date, no quantitative biochemical determinations have been made of the relative abundance of different NMDAR populations in intact hippocampus, the region extensively correlated with NMDAR-dependent long-term potentiation. We investigated subunit composition and subunit-specific interactions in CA1/CA2 of rat hippocampus. Using sequential immunoprecipitations to deplete either NR2B or NR2A, di-heteromeric NR1/NR2A and NR1/NR2B receptor populations were isolated from postnatal day 7 (P7) hippocampus and P42 and 6-month-old CA1/CA2. Quantitative Western blot analysis revealed that 60-70% of NR2A and 70-85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes. Isolated di-heteromeric receptor fractions were used to examine NR2A- or NR2B-specific interactions with synapse-associated proteins. Our results indicate that NR2A- or NR2B-containing NMDARs associate similarly with postsynaptic density-95 (PSD-95), synapse-associated protein 102, and PSD-93 at P42. However, NR2A-containing receptors coimmunoprecipitated a greater proportion of the synaptic proteins neuronal nitric oxide synthase, Homer, and beta-catenin. Finally, mass spectrometry analysis of isolated di-heteromeric receptors identified a novel NMDAR interactor, collapsin response mediator protein 2, which preferentially associates with NR2B-containing di-heteromeric NMDARs. In summary, in rat hippocampus, NR2A and NR2B exist primarily in di-heteromeric complexes that interact similarly with PSD-95-related proteins but are associated with different protein complexes.

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.

Regulated exocytosis is essential for many biological processes and many components of the protein trafficking machinery are ubiquitous. However, there are also exceptions, such as SNAP-25, a neuron-specific SNARE protein that is essential for synaptic vesicle release from presynaptic nerve terminals. In contrast, SNAP-23 is a ubiquitously expressed SNAP-25 homolog that is critical for regulated exocytosis in non-neuronal cells. However, the role of SNAP-23 in neurons has not been elucidated. We found that SNAP-23 was enriched in dendritic spines and colocalized with constituents of the postsynaptic density, whereas SNAP-25 was restricted to axons. In addition, loss of SNAP-23 using genetically altered mice or shRNA targeted to SNAP-23 led to a marked decrease in NMDA receptor surface expression and NMDA receptor currents, whereas loss of SNAP-25 did not. SNAP-23 is therefore important for the functional regulation of postsynaptic glutamate receptors.